Prior to the present invention, it had long been recognized that enterobacterial lipopolysaccharides (LPS) was a highly potent stimulator of the immune system. A variety of responses, both beneficial and harmful, can be elicited by sub-microgram amounts of this substance. The fact that some of these responses are harmful, and can in fact be fatal, has to date precluded clinical use of LPS per se. It is now also well-appreciated that the endotoxic activites associated with bacterial lipopolysaccharides (LPS) reside in the lipid A component of LPS.
Accordingly, much effort has been expended towards attenuating the toxic attributes of lipid A and LPS without diminishing their beneficial immunostimulatory activities. Notable among these efforts was that of Edgar Ribi and his associates, which resulted in the production of a derivative of lipid A referred to originally as refined detoxified endotoxin (RDE) but more recently as monophosphoryl lipid A (MPL). MPL is produced by refluxing LPS (or lipid A) obtained from heptoseless mutants of gram negative bacteria (e.g. Salmonella sp.) in mineral acid solutions of moderate strength (e.g., 0.1N HCl) for a period of approximately 30 minutes. This treatment results in the loss of the phosphate moiety at position 1 of the reducing-end glucosamine. Coincidentally, the core carbohydrate is removed from the 6' position of the non-reducing glucosamine during this treatment. The result is the monophosphoryl derivative of lipid A, MPL. The structure of MPL is shown below: ##STR1##
MPL exhibits considerably attenuated levels of the endotoxic activities normally associated with lipid A and LPS, such as pyrogenicity, local Shwarzman reactivity, and toxicity in the chick embryo 50% lethal dose assay (CELD.sub.50). It retains the ability of lipid A and LPS, however, and to, among other things, act as an adjuvant.
The difficulty with this method of detoxifying LPS and lipid A is that it invariably results in the loss of the core moiety attached to position 6' of the non-reducing glucosamine. This is significant since the core region is highly conserved among LPS's obtained from different genera of Enterobacteriaciae; immunity against the core region is therefore protective against a wide variety of gram negative bacterial challenges. This was demonstrated by the work of Ziegler et al. (New Eng. J. Med. 307, 1225: 1982), for example.
Considerable benefits would accrue from being able to immunize individuals against enterobacterial LPS, as evidenced by the fact that approximately 90,000 deaths occur annually from gram negative sepsis and associated endotoxemia. At the present time, however, it is only possible to immunize with fully toxid LPS, since detoxification by acid hydrolysis results in loss of the core region.
Alkaline hydrolysis has also been used in the past to detoxify LPS, but the conditions which have generally been used result in complete saponification of the lipid A moiety. This, of course, not only reduces the endotoxicity of the starting LPS, but also eliminates the other, more beneficial, activities as well. Furthermore, such treatment also reduces the immunogenicity of LPS, since it is essentially converted by this treatment into a polysaccharide antigen with no amphipathic character. In general, however, none of the early references teach that removal of one particular fatty acid from lipid A would render it non-toxic, while not affecting its immunostimulating activities.
Accordingly, one or more of the following objects will be achieved by the practice of this invention. It is an object of this invention to provide modified lipopolysaccharrides and, in particular, modified lipid A. Another object of this invention is to provide a modified lipid A which retains the core moiety attached to the 6' positions of non-reducing glucosamine. A further object of the present invention is to provide a modified lipid A which retains the core moiety and accordingly, protection against a wide variety of gram negative challenges. A still further object of the present invention is to provide a process for the preparation of the modified lipid A which renders it less endotoxic without adversely affecting their antigenic or immunostimulating properties. Another object is to provide pharmaceutical compositions containing the modified lipopolysaccharides and a method for their use. These and other objects will readily become apparent to those skilled in the art in light of the teachings herein set forth.